1. Field of the Invention
The present invention relates to a novel way of running tests to determine the presence of particular nucleic acid sequences in test samples and to novel probes useful therefor.
2. Background Information
The application of two non-overlapping DNA probes for hybridization has been disclosed in PCT patent application No. 83/01459, European patent application Nos. 0070687 and 0070685. PCT No. 83/01459 and 0070687 disclose the application of two non-overlapping hybridization probes for the detection of a particular polynucleotide sequence in a test sample. One of the probes is fixed to a solid support prior to hybridization. Although this method eliminates the problem of electrophoretic separation of nucleic acids before hybridization, the process is slow because of the heterogeneous phases utilized.
European publication No. 0070685 discloses a homogeneous phase two probe assay with a non-radiative transfer method. This method needs sophisticated equipment to monitor hybridization. The background cannot be eliminated because of brownian motion, some nonspecific reactions, and because the concentration of the unhybridized probes present in solution is always very high compared to the hybridized probes.
A heterogenous system involving two probes, one of which is immobilized, is described in application Ser. No. 815,694, filed Jan. 21, 1986, now abandoned in favor of application Ser. No. 07/052,634, filed May 20, 1987 and Ranki et al, Gene, 21, 77-85, (1983). The probes can be DNA, RNA, mixed nucleic acids or oligonucleotides. There are disclosed tests for particular nucleic acid sequences, such as that indicating sickle cell anemia, for example, by contacting the sample with two probes. The immobilized probe, otherwise identified as a separation probe, is immobilized on a support such as nitrocellulose. The other probe, identified as the detection probe, carries a label for ultimate assay. Both probes include different nucleic acid fragments, both complementary to a different portion of the test sequence if present in the test sample. The sample and probes are mixed, subjected to hybridizing conditions and, if the sample contains the right sequence, its nucleic acid will serve as a bridge between the two probes. Thereby the label of the labeled probe will become attached to the solid support. The support is removed and then "read" for the presence of the label.
The probes can be such that the label on the solid support will indicate either a positive or negative result with regard to the condition to be detected. In addition to sickle cell anemia, the test can be for any other genetic condition, e.g., thalassemia, Tay-Sachs, etc. An identical procedure can also be followed for the detection of bacteria or viruses in test samples.
While such process produces satisfactory results, it was desired to speed up the diagnostic process, without the disadvantages attending the homogeneous two probe assay noted hereinabove.
A homogeneous system involving two probes has been described in patent application Ser. No. 704,130, filed Feb. 21, 1985, now pending. This method uses two non-overlapping probes, one of which is labelled for detection and the other for the separation of the hybrid. The assay takes place in a homogeneous solution and the hybrid is subsequently separated by an immobilization reaction with a solid support and the separation probe.
Australian Patent Specification 40,310/85 concerns the use of an azide group to label probes. The Australian Patent Specification discloses the use of two probes involved in a single assay.